Compositions and methods for treating angiogenesis-related diseases, wounds and cosmetic use of components of Angelica sinensis, and methods of preparation thereof

ABSTRACT

Compositions and methods for treating angiogenesis-related diseases and for skin care in mammals is disclosed that includes, as an active pharmaceutical agent, an effective amount of purified extract from  Angelica sinensis , or a fraction or a lyophilizate thereof, or one or more active component contained in said extract.

CROSS-REFERENCES TO RELATED APPLICATIONS

This application claims priority to provisional U.S. patent applicationSer. No. 60/505,174, filed Sep. 22, 2003, entitled, “Compositions andMethods for Treating Angiogenesis-related diseases, Treating Wounds andCosmetic Use from Herb Extracts from Angelica sinensis, and Methods ofPreparation thereof”, which is incorporated herein by reference in itsentirety.

BACKGROUND OF THE INVENTION

Angiogenesis (the growth or assembly or formation of blood vessels) isan essential process during embryogenesis and pre-adult physiologicaldevelopment. In adults, angiogenesis is an important beneficialreparative process in wound healing, menstrual cycle, and bone repair,as well as in the recovery from stroke, peptic ulcers and certaincardiovascular diseases. Preservation of healthy network of dermal bloodvessels is also essential for skin health and delaying skin ageingprocess. Several endogenous angiogenesis stimulators or factors havebeen reported that include basic fibroblast growth factor (bFGF) and itsfragments, vascular endothelial growth factors (VEGF) and theirfragments, interleukin 1 alpha and others. Therapeutic administration ofsome of these factors has been described to be useful and beneficial inmany of the aforementioned physiological conditions. For example, basicfibroblast growth factor (bFGF) has been reported to improve quality oflife as assessed by Seattle Angina Questionnaire and improve exercisetolerance as assessed by treadmill exercise testing in patients ischemicheart disease not amenable to treatment with coronary artery bypassgraft (CABG) or percutaneous transluminal coronary angioplasty (PTCA)(Laham et al., 2000). Moreover, bFGF was shown by magnetic resonanceimaging (MRI) to increase regional wall thickening and to reduce theextent of the ischemic area at all time points compared with baseline.However, effective angiogenesis stimulators with low side effect stillrepresent a therapeutic gap even today, since there are no angiogenesisstimulators available that are approved by FDA for broad human use.

Furthermore, conditions, such as wounds, photodamaged and aged skinrequire activation of proliferation of several cell types, includingendothelial cells and fibroblasts.

In our invention, we teach that an aqueous extract from Angelicasinensis or a fraction or a lyophilizate thereof, or one or more activecomponents contained in said extract, stimulates endothelial cell growthin vitro, vessel-like structure formation in vitro and angiogenesis invivo, indicating that Angelica sinensis or the compositions thereof maybe useful for treating a vast array of pathologic conditions whichrequire stimulation of angiogenesis, such as cardiovascular diseases,stroke, bone loss, peptic ulcers and skin wounds. It also indicates thatSBD.4 may delay the process of dermal ageing by maintaining healthyirrigation of the skin and find use in the cosmetic products as activeingredients.

Furthermore, we teach that SBD.4 stimulates the proliferation of dermalfibroblasts. Proliferation of dermal fibroblasts is necessary for woundclosure. Several angiostimulators were shown to enhance fibroblastgrowth (e.g. bFGF). Fibroblast cells are the major component of thedermis layer of the skin. They secrete collagens and other extracellularmatrix components, which determine the elasticity, thickness andresilience of the skin. Therefore, natural products, which can supportfibroblast metabolism and survival, but which at least minimallystimulate cancer cells, are of major interest as, for example, woundhealing and cosmetic active ingredients.

Furthermore, we teach that SBD.4 not only protects and enhancesmicrocirculation and dermal fibroblast growth, but also stimulatescollagen I levels in fibroblast culture medium. Collagen I is a keycomponent of extracellular matrix in the skin. In aged and damaged skin,collagen I levels are decreased due to the lower amount and quality ofdermal fibroblasts. Collagen I output is also an important part of thewound healing process.

SUMMARY OF THE INVENTION

This invention describes a new use of a water extract (also referredhere to as crude aqueous extract) from the roots of Angelica sinensis(family of Umbelliferae; also known as dang gui, dan gui, dong quai,tang kuei, Chinese angelica) or a fraction or a lyophilizate thereof, orone or more active components contained in said extract, this use beingto stimulate endothelial cell proliferation, angiogenesis, andfibroblast growth. The abovementioned aqueous extract or a fraction or alyophilizate thereof, or one or more active components contained in saidextract which stimulate endothelial cell proliferation, angiogenesis,and fibroblast growth, also referred here to as SBD.4, or SBD.4bioactive material is also known as ProDermin and Angiogent.

The stimulation of angiogenesis and fibroblast growth by SBD.4 may be avaluable treatment for cardiovascular and other ischemic diseases, boneloss, stroke, peptic ulcers, wounds, for personal care (such as skin,lip, hair care), and for other conditions where the stimulation offibroblast growth and/or angiogenesis, or protection of vascularnetworks is beneficial.

BRIEF DESCRIPTION OF THE INVENTION

FIG. 1. Stimulatory effect of SBD.4 purified by the means ofDEAE-cellulose on proliferation of bovine endothelial cells, as comparedwith a potent angiogenesis stimulator bFGF (1 ng/ml).-bFGF illustratesBCE proliferation in the absence of bFGF and SBD.4. The finalconcentrations of SBD.4 tested are 0.5 μg/ml, 2 μg/ml and 10 μg/ml.

FIG. 2. Stimulatory effect of HPLC-purified SBD.4 on the proliferationof BCE (bovine capillary endothelial) cells, as compared with a potentangiogenesis stimulator bFGF, at different conditions. SBD.4 was testedon the proliferation of bovine capillary endothelial (BCE) cells in 96well tissue culture plate, according to Skehan et coll. (1990). Cellnumber was estimated after 72 h of exposure to experimental treatments,and represented as optical density (OD) absorption of cell-associatedsulforhodamine B staining at 570 nm. “(RT)” designates bFGF (1 ng/ml) orSBD.4 (2000 ng/ml) preincubated at room temperature for 7 days beforeactivity test. “Pronase” signifies that the sample was preincubated withpronase (100 ug/ml in the presence of calcium) for 3 h at 37° C., beforeactivity test. The dotted line indicates the baseline level of cellproliferation in the absence of any growth factors (Control). Error barsrepresent standard deviation. Number of tests (n)=3.

FIG. 3. Tridimensional tube formation assay, in vitro. A: Capillaryendothelial cells form a monolayer in the absence of a remodelingstimulus. B: SBD.4 (5 μg/ml, purified by HPLC) induces remodeling of themonolayer that culminates in a tridimensional network of capillarystructures. C: Remodeling response in the same model, induced by bFGF(10 ng/ml).

FIG. 4. Stimulation of angiogenesis by SBD.4 (50 micrograms/milliliter,3 kD cut off filtration-purified) in the CAM assay (each panel is areproduction of a color photograph). A: A vascular zone (in the middleof the panel), before putting the pellet. B: Same zone, 24 h later. Notea new blood vessel passing right through the pellet. C: A differentexperiment, showing a typical “spokes of a wheel” angiostimulatoryeffect 48 h after placement of the SBD.4 pellet.

FIG. 5. Effect of SBD.4 (DEAE and HPLC-purified) on the proliferation ofhuman dermal fibroblasts (HDF) and on the type I collagen in the mediumconditioned by these cells. A: SBD.4 stimulates the proliferation ofHDF. B: SBD.4 increases type I collagen levels in the HDF-conditionedmedium, as measured by sandwich ELISA, according to Dobak and coll.(1994). Note that SBD.4 has stronger effect on collagen output andfibroblast proliferation than 5 ng/ml fibroblast growth factor (positivecontrol). Error bars represent standard deviation. Number of tests(n)=3.

FIG. 6. Wound closure stimulated by SBD.4 (3 kd cut-off filtered aqueousextract) as compared with PBS (negative control) and becaplermin(positive control), in genetically diabetic mice. The stimulatory effectof SBD.4 was statistically significant while the effect of becaplerminwas not meaningfully different from the control, as calculated byone-way analysis of variance (ANOVA). Each data point represents mean+/−SEM. n=12 for Control and n=6 for SBD.4 and becaplermin.

FIG. 7. Composition of SBD. 4 at different levels of purification asdefined by high pressure liquid chromatography-coupled mass spectrometry(HPLC-MS). Y axis represents the intensity of the signal, X axisrepresents elution time for panels A, C, D and mass-to-ion ratio (m/z)+1 (reflecting the molecular mass) for panels B, D, F. HP LC-MSconditions were: column flow: 0.5 ml/min; stoptime (run time): 10 min 30sec; solvent conditions: Start: 95% solvent A (99.96% H2O-0.04% formicacid)/5% solvent B (100% acetonitrile) Finish (at time 9 min): 90%solvent B/10% solvent A. The HPLC column was Reverse Phase C18 Synergypolar RP (Phenomenex, Torrance, Calif.) and the program was Polar-2Agilent Easy Access Method. Further details are available on the panels.A-B: Composition SBD.4 purified by extracting the dry roots of Angelicasinensis (cut in pieces of approximately 0.5 cm×0.5 cm) in water at 25°C. for 20 min, filtered through 0.22 micron filter, then filteredthrough a 3 kilodalton (kD) cutoff centrifuge filter (Millipore). Thethree UV absorption profiles selected from the diode array spectrum ofthe SBD.4 eluate in panel A are (from top to bottom) at 254 nm(nanometer), 225 nm, 350 nm. The fourth panel represents the massspectrometry (MS) profile of the HPLC eluate. The panel B represents aspecific region of interest eluted from the column between 1.786 and2.048 min. C-D: Composition of SBD.4 initially purified as mentioned inthe description of panels A-B above, then further purified by one cycleof reverse phase C18 HPLC chromatography under the conditions describedin the section “Methods of Preparation and Characterization of SBD.4”.The three UV absorption profiles selected from the diode array spectrumof the SBD.4 eluate in panel C are (from top to bottom) at 254 nm, 225nm, 350 nm. The fourth panel represents the MS profile of the HPLCeluate. MS analysis in panel D represents the region of interest elutedbetween 1.786 and 2.103 min. E-F: Composition of SBD.4 purified asmentioned in the description of panels A-B, then further purified byDEAE-cellulose (Pharmacia) chromatography as follows: one volume ofSBD.4 solution was loaded on a DEAE-cellulose column of equivalentvolume and the column was washed by 3 column volumes of H₂O. SBD.4 waseluted by 3 volumes of NH₄OH pH 8 followed by 2 volumes of H₂O. The flowrate for a column of 2.5 cm diameter and 20 cm high was 2 ml/minthroughout the whole DEAE-cellulose purification process. These 5 elutedvolumes were pooled and lyophilized. The lyophilizate was dissolved inwater in one fiftieth of the original volume of the pooled eluate, andmethanol was added up to 75%. After homogenization, the solution wascentrifuged and supernatant was collected, vacuum-dried, redissolved inwater and purified on HPLC column, as detailed in the description ofpanels C-D. The four UV absorption profiles selected from the diodearray spectrum of the SBD.4 eluate in panel E are (from top to bottom)at 225 nm, 254 nm, 350 nm and 280 nm. The fifth panel represents the MSprofile of the HPLC eluate. The bottom graph on panel E represents ionextraction at 174 of the total ion current, showing the presence of asmall amount of the 174D entity in this fraction. MS analysis in panel Frepresents the region of interest eluted between 1.749 and 1.861 min.

FIG. 8. Flow chart of Angelica sinensis component purification andcharacterization.

DETAILED DESCRIPTION OF THE INVENTION

Methods of Preparation and Characterization for SBD.4

The description here is to illustrate, not to limit, methods used toprepare SBD.4 (FIG. 8). First, SBD.4 is obtained by extracting the rootsof Angelica sinensis in water, to provide the aqueous extract. Roots areplaced in water at 5% (w:v) and brought to boil. The typical boilingtime is 30 minutes and the typical extraction temperature is 98 degreeCelsius. However, it is reasonable to assume that lower or higher ratioof roots:water, shorter or longer times of extraction as well as loweror higher temperature of extraction will result in the extraction of thesame angiostimulatory material. For example, extraction at roomtemperature results in similar activity than extraction at highertemperatures. Also, it is reasonable to assume that the same SBD.4angiostimulatory activity may be extracted with a solution combiningwater and alcohol including, but not limited to, methyl or ethylalcohol, or another polar solvent.

Following aqueous extraction, the extract mentioned above is centrifugedat the speed of about 2,000 g to remove insoluble particles, thensterile-filtered through a Nalgene (Rochester, N.Y.) 0.22 micron porefilter. Alternatively, the extract can be filtered through a set offilters of decreasing pore size, culminating with the filtration through0.22 micron pore size filter. Pectinase can be added to the extract toimprove its flow rate through filtering devices by digesting certainpolysaccharides in the extract, without subsequent loss of biologicalactivity of SBD.4. Furthermore, the aforementioned extract can befiltered through a centrifugal filter device of cut-off molecular weightof 10,000 daltons (Millipore, Bedford, Mass.) without loosing theactivity mentioned in examples 1-7. The SBD.4 angiostimulatory materialis collected in the lower chamber of the centrifugal filter device(Millipore. Bedford, Mass.), suggesting that the molecular weight ofSBD.4 is less then 10,000 daltons (D). Furthermore, the extract can befiltered through a centrifugal device of three 3,000 D molecular weightcut-off (Millipore), and the angio- and fibroblast stimulatory materialis collected in the lower chamber, suggesting that the molecular weightof this angiostimulatory material is 3,000 daltons or less. Such 3,000 Dcut off filter-purified SBD.4 has been used in Example 3, 5 and 6 (FIG.6 and Tables I and II) and in Example 8 (FIG. 7A-B). Furthermore, theextract can be subjected to G10 Sepharose (Sigma, St. Luis)chromatography, which allows to separate compounds of molecular weightlower than 700 daltons from other components of the mix. When subjectedto such chromatography, SBD.4 activity is absent from the fractioncorresponding to the void column volume and from immediately subsequentfraction, which contains compounds of molecular weight higher than 700daltons. On the contrary, SBD.4 is present in the third and fourthfraction (all fractions are of equal volume), which contains moleculesof molecular weight lower than 700 daltons.

SBD.4 angiostimulatory material can be further purified by using theDEAE-cellulose chromatography (Pharmacia), whereas the cell stimulatoryactivity binds to DEAE-cellulose. Endothelial cell andfibroblast-stimulatory activity can be recovered from this resin by acidelution including, but not limited to 100 mM acetic acid and 100 mMhydrochloric acid, or weak alkaline elution such as NH₄OH of pH about 8.Fibroblast-stimulatory activity can be recovered by the aforementionedconditions as well as by strong alkaline elution (100 mM NaOH). SBD.4purified by loading on DEAE-cellulose, washing with water and elutingwith NH₄OH pH about 8, can be further purified by precipitation with 75%methanol, centrifugation, lyophilization of the supernatant,redissolution in water and purification by high pressure liquidchromatography (HPLC) on C18 reverse phase Targa (Higgins Analytical,Sunnyvale, Calif.) column. Lower or higher concentrations of methanolcan be used instead of the abovementioned 75%. The use of lowerconcentrations of methanol will result in the precipitation of fewerimpurities, while at least a fraction of SBD.4 will remain in thesupernatant precipitated with up to about 98% methanol. The activematerial (i.e. material, which stimulates endothelial and fibroblastcell proliferation) elutes relatively early (at ˜7.5 min, sign ˜ meaning“approximately” throughout this application) under the experimentalconditions as follows: column dimensions: length: 250 mm; diameter: 10mm; particle size: Smicrons. Sample is injected in 2 ml water, eluted inwater (flow rate: 2 ml/min). Elution is monitored by refractometry andUV absorption at 245 nm. The HPLC-coupled mass spectrometry (HPLC-MS)analysis shows that one of the components in the active fraction is amolecular entity of 218 daltons. A smaller quantity of an entity of 174daltons was also present (see Example 8, FIG. 7E-F). Such DEAE andHPLC-purified SBD.4 has been used in Example 4 (FIG. 5) and Example 8(FIG. 7E-F).

In Example 2 (FIG. 2), Example 7 (Table III) and Example 8 (FIG. 7C-D)SBD.4 was purified as following: roots of Angelica sinensis arewater-extracted and filtered through 0.22 micron filter, as describedabove. The extract was then filtrated through a 5000 daltons molecularcut-off Pellicon XL 50 Biomax filter (Millipore) and subjected to HPLCchromatography on reverse phase C18 Targa (Higgins Analytical,Sunnyvale, Calif.) column. The active material (i.e. material, whichstimulates endothelial and fibroblast cell prolifertion) elutedrelatively early (at ˜7.5 min). The experimental conditions were asfollows: column dimensions: length: 250 mm; diameter: 10 mm; particlesize: 5 microns. Sample was injected in 2 ml water, eluted in water(flow rate: 2 ml/min). Elution was monitored by refractometry and UVabsorption at 245 nm.

Furthermore, SBD.4 can be also purified by aforementioned waterextraction and filtration, followed by sequential methanol precipitationand high pressure liquid chromatography (HPLC) on C18 reverse phasecolumn, as described below: Roots of Angelica sinensis werewater-extracted and filtered through 0.22 micron filter, as describedabove. The extract was then filtrated through a 5000 daltons molecularcut-off Pellicon XL 50 Biomax filter (Millipore). The filtrate (12 gsolid solubilized in 400 ml water) was collected and concentrated undervacuum in a rotovap to about 25 ml. Three hundred ml of methanol wereadded, stirred overnight and precipitated solids (Fraction 1, about 3.7g) were separated from the supernatant by centrifugation. Thesupernatant was dried in rotovap, 3.2 g of the dried material wasdissolved in 18 ml of water and 3 ml of methanol were added. Theresulting precipitate (Fraction 2) was separated from the supernatant,the supernatant was evaporated, dissolved in 9 ml of water and 3 ml ofmethanol were added. The resulting precipitate (Fraction 3) wasseparated by centrifugation and the supernatant was dried andredissolved in 3 ml water, to which 3 ml of methanol were added. Afterremoving the precipitated material, the supernatant was dried and itsweight was estimated at 1.2 g (Fraction 4). The bioactivity of thefractions 1-4 was compared (by measuring its endothelial cellstimulatory activity, as in example 1), and Fraction 4 was found to bethe most active. SBD.4 can be lyophilized and redissolved in waterwithout loosing the aforementioned endothelial and fibroblast cellstimulatory activity.

SBD.4 is an aqueous extract from roots of Chinese medicinal herbAngelica sinensis. Preferably, SBD.4 is a molecule or an extract ofmolecules of molecular weight of ten thousand daltons or less. Morepreferably, it is a molecule or an extract of molecules of molecularweight of three thousand daltons or less. Most preferably, it is amolecule or an extract of molecules of 700 daltons or less.

SBD.4 is heat-stable. Angiostimulatory activity of SBD.4 is maintainedeven after boiling for 30 minutes in water at ninety five degreescentigrade.

SBD.4 binds to diethylaminoethyl cellulose (DEAE) cellulose ion—exchangeresin and to reverse-phase resins, and is recovered by selected elutionbuffers.

SBD.4 is stable at pH 7. More preferably, SBD.4 is stable at a pH rangeof at least 3-8

SBD.4 can be acetylated with the use of pyridine and acetic anhydride (amethod useful for acetylation of sugars and aminoacids), which indicatesthe presence of hydroxyl and/or amino group(s). Because at roomtemperature and under base treatment the acetylation of SBD.4 can bereversed only partially, it is assumed that both amino group(s) andhydroxy group(s) are present in SBD.4.

SBD.4 is very polar, because it elutes relatively early (at ˜7.5 min)from Targa C18 HPLC reverse phase column. Column dimensions are: length:250 mm; diameter: 10 mm; particle size: 5 microns. Sample is injected in2 ml water, eluted in water (flow rate: 2 ml/min). SBD.4 may beadministered to humans in various forms such as tablets, hard gelatincapsules, or liquid preparations, as oral medicines, wound dressings,topical drugs or skin care products, or injectable drugs.

The above-described methods are useful in obtaining active componentsfrom Angelica sinensis for the following application. However, thedescription is not meant to restrict the use in Angelica sinensisspecies only, the genus Angelica including but not limited to Europeanand American Angelica species, or sometimes the family Umbelliferae iswithin the scope of the present invention.

Pharmaceutical Appications

The present invention provides a pharmaceutically acceptable formulationof SBD.4- a water extract (also referred hereto as crude aqueousextract) from the roots of Angelica sinensis, a fraction or alyophilizate thereof, or one or more active components contained in saidextract, useful in the methods of the present invention. In oneembodiment, SBD.4 is packaged in a sachet that is decanted into apotable liquid for oral administration to the patient. In thisembodiment, the liquid can be a syrup or, more conveniently, a commonlyconsumed liquid, such as water, fruit juice, or cola. In anotherembodiment, the Angelica sinensis extract is formulated as a tablet orpill. In yet another embodiment, SBD.4 is formulated in a medicaldevice, such as, but not limited to a wound dressing. A decidedpractical advantage of the compounds of the present invention is thatthe compounds can be administered in any convenient manner such as bythe oral, intravenous, intramuscular, topical, or subcutaneous routes.

Thus, Angelica sinensis extracts can be orally administered, forexample, with an inert diluent or with an assimilable edible carrier,including, but not limited to a power bar, or it can be enclosed in hardor soft shell gelatin capsules, or compressed into tablets, orincorporated directly with the food of the diet. For oral therapeuticadministration, Angelica sinensis extracts can be incorporated withexcipients and used in the form of ingestible tablets, buccal tablets,troches. capsules, elixirs, suspensions, syrups, wafers, and the like.Such compositions and preparations contain enough of the active agent todeliver the therapeutically active doses described above. In someembodiments, the formulation will be glucose-free.

The tablets, troches, pills, capsules, and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch, orgelatin; excipients such as dicalcium phosphate; a disintegrating agentsuch as corn starch, potato starch, alginic acid, and the like; alubricant such as magnesium stearate; a sweetening agent such assaccharin; and/or a flavoring agent such as peppermint, oil ofwintergreen, or cherry flavoring. When the dosage unit form is acapsule, it can contain, in addition to materials of the above types, aliquid carrier. Various other materials can be present as coatings or tootherwise modify the physical form of the dosage unit. For instance,tablets, pills, or capsules can be coated with shellac. A syrup orelixir can contain the active compound, a sweetening agent, methyl andpropylparabens as preservatives, and a flavoring such as cherry ororange flavor. Of course, any material used in preparing any dosage unitform should be pharmaceutically pure and substantially non-toxic in theamounts employed. In addition, the active compound can be incorporatedinto sustained-release preparations and formulations. The Angelicasinensis extract can also be administered parenterally orintraperitoneally. A solution of the active compound as a free acid orpharmacologically acceptable salt can be prepared in water suitablymixed with a surfactant such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols and mixturesthereof, and in oils. Under ordinary conditions of storage and use,these preparations contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include sterileaqueous solutions or dispersions and sterile powders for theextemporaneous preparation of sterile injectable solutions ordispersions. In all cases, the form must be sterile and, in final form,must be fluid to the extent that easy syringability exists. It must bepreserved against the contaminating action of microorganisms such asbacteria and fungi.

The pharmaceutical forms suitable for topical use include oil and wateremulsions and liposomal formulations, as well as lotions, creams,carriers and ointments commonly used for topical administration ofdrugs, including, but not limited to carboxymethylcellulose,poly-lactic/glycolic acid, hydroxypropyl/methylcellulose,carboxymethylcellulose foam, hydroxyapatite or hyalouronic gel.

SBD.4 may be also useful for the treatment of wounded, damaged orotherwise fragilized skin, like the diabetic skin under the form ofaqueous soaking solution.

The carrier can be a solvent or dispersion medium containing, forexample, water, ethanol, polyol, for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like, suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating such as lecithin, by the maintenance ofthe required particle size in the case of dispersion and by the use ofsurfactants. The prevention of the action of microorganisms can bebrought about by various anti-bacterial and anti-fungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example sodium chloride. Prolonged absorption of theinjectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions are prepared by incorporating the activecompound in the required amount in the appropriate solvent with variousother ingredients enumerated above, as required, followed by filteredsterilization. Generally, dispersions are prepared by incorporating thevarious sterilized active ingredient into a sterile vehicle whichcontains the basic dispersion medium and the required other ingredientsfrom those enumerated above. In the case of sterile powders for thepreparation of sterile injectable solutions, the preferred methods ofpreparation are vacuum drying and the freeze drying technique, whichyield a powder of the active ingredient plus any additional desiredingredient from previously sterile filtered solution thereof.

As used herein, a “pharmaceutically acceptable carrier includes any andall solvents, dispersion media, coatings, antibacterial and antifungalagents, isotonic agents, absorption delaying agents, and the like. Theuse of such media and agents for pharmaceutical active substances iswell known in the art. Except insofar as any conventional media or agentis incompatible with the active ingredient, its use in the therapeuticcompositions of the invention is contemplated. Supplementary activeingredients can be incorporated into the compositions of the invention.

It is essentially advantageous to formulate parental and othercompositions in dosage unit form for ease of administration anduniformity of dosage. Dosage unit form as used herein refers tophysically discrete units suited as unitary dosages for the mammaliansubjects to be treated; each unit containing a predetermined quantity ofactive material calculated to produce the desired therapeutic effect inassociation with the required pharmaceutical carrier.

Another aspect of the invention includes but is not limited to,promoting angiogenesis in tissue by directly contacting the tissue withSBD.4 in an amount effective to promote angiogenesis in said tissue. Oneembodiment of the invention is promoting angiogenesis in a tissueselected from the group consisting of endothelial, cardiac, cerebral,muscular, vascular, transplanted, dermal, epidermal or wounded. Anotherembodiment is that the tissue could also be ischemic wherein theischemic tissue is selected from the consisting of myocardial ischemictissue, cerebral ischemic tissue, veno-occlusive diseased tissue,wounded tissue, damaged dermal tissue, damaged epidermal tissue,diabetic tissue and myocardial ischemic tissue wherein said myocardialischemic tissue is implicated in coronary artery diseases.

The stimulation of angiogenesis and fibroblast growth by SBD.4 from herbextract of Angelica sinensis may be a valuable treatment for ischemicdiseases, such as, but not limited to cardiovascular and peripheralischemic diseases, stroke, peptic ulcers, wounds, diabetic skin andother diabetic tissues, for bone regeneration, for personal care (suchas skin, lip, hair care), and for other conditions where the stimulationof fibroblast growth and angiogenesis, or protection of vascularnetworks is beneficial.

The pharmaceutical formulation methods described above can be similarlyapplied to the following wound-healing application.

Wound-Healing Applications

The present invention provides improved methods for promoting woundhealing and reducing scar formation. In particular, an aqueous extractfrom the roots of Angelica sinensis or a fraction or a lyophilizatethereof, or one or more active components contained in said extract,provide significant improvements over the prior art method of promotingwound or reducing scar formation.

As used herein, the term “wound” is used throughout the specification todescribe skin wounds, which are treated by the formulations and themethods, described herein as well as tissue wounds. A skin wound isdefined herein as a break in the continuity of skin tissue that iscaused by direct injury to the skin. Several classes includingpunctures, incisions, excisions, lacerations, abrasions, atrophic skin,or necrotic wounds and bums generally characterize skin wounds. Thecompositions and methods of the invention are useful for enhancing thehealing of all wounds of the skin. In particular, the present inventionprovides methods and compositions suitable for treatment of wounds indiabetics, normal patients and surgical patients.

A “tissue wound” as used herein is a wound to an internal organ, such asa blood vessel, intestine, colon, etc. The materials of the inventionare useful for enhancing the wound healing process in tissue woundswhether they arise naturally or as the result of surgery. For instance,during the repair of arteries the vessel needs to be sealed and woundhealing must be promoted as quickly as possible. The compositions of theinvention can speed up that process. The compositions of the inventionare also particularly useful for the treatment of damaged tissues in thedigestive system.

The methods of the invention are also useful for preventing scarformation. The compositions can be used to prevent the formation of ascar at the same time as promoting wound healing. Alternatively, thecompositions may be used for preventing scar formation by reducing orinitiating regression of existing scars. Scar tissue as used hereinrefers to the fiber rich formations arising from the union of opposingsurfaces of a wound.

The compositions and methods of the invention may also includeadditional therapeutic and/or pharmacologically acceptable agents. Forinstance, the compositions or methods may involve other agents for thetreatment of wounds such as, for instance, dexpanthenol, growth factors,enzymes or hormones, povidon-iodide, fatty acids, such as cetylpyridinium chloride, antibiotics, and analgesics.

Growth factors include, but are not limited to, fibroblast growthfactors (FGF), FGF-1, FGF-2, FGF-4, thymosins, platelet-derived growthfactors (PDGF), insulin-binding growth factors (IGF), IGF-1, IGF-2,epidermal growth factor (EGF), transforming growth factor (TGF),TGF-.alpha., TGF-.beta., cartilage inducing factors-A and -B,osteoid-inducing factors, osteogenin, bone morphogenic proteins, andother bone growth factors, collagen growth factors, heparin-bindinggrowth factor-1 or -2, and their biologically active derivatives. Thecompositions may also include antiseptics.

Cosmetic Applications

An aqueous extract from Angelica sinensis or a fraction or alyophilizate thereof, or one or more active components contained in saidextract, may be utilized in the formulation of cosmetic compositionsbecause of its novel properties in not only protecting and enhancingmicrocirculation and dermal fibroblast growth, but also stimulatingcollagen I levels and fibroblasts.

Cosmetic compositions are preparations applied to the surface of thebody for the purpose of enhancing its appearance. These compositions canbe make-up preparations, applied to bring about temporary effects,lasting only so long as the preparations remain on the body surface, ortreatment preparations, which effect no immediately noticeable changebut which, after repeated use, are expected to have a beautifyingeffect.

The cosmetic compositions of the present invention may be skin careproducts such as lotions, creams, shampoos, cleansers, make-upfoundations, footbaths, etc. The compositions of the invention may beemulsions of liquid or semi-liquid consistency of the milk type,obtained by dispersion of an oil phase in an aqueous phase or viceversa; or suspensions or emulsions of soft consistency of the creamtype.

All oils used in the production of cosmetic compositions are suited foruse in the compositions of the present invention. There may be mentionedhydrocarbons such as mineral oils, petrolatum and squalane; animal andvegetable triglycerides such as almond oil, peanut oil, wheat germ oil,linseed oil, jojoba oil, oil of apricot pits, oil of walnut, oil of palmnuts, oil of pistachio nuts, oil of sesame seeds, oil of rapeseed, cadeoil, corn oil, peach pit oil, poppyseed oil, pine oil, borage oil,castor oil, soybean oil, Lycium barbarum oil, avocado oil, saffloweroil, coconut oil, hazelnut oil, olive oil, grapeseed oil, and sunflowerseed oil; hydroxy-substituted C.sub.8-C.sub.50 unsaturated fatty acidsand esters thereof; C.sub.1-C.sub.24 esters of C.sub.8-C.sub.30saturated fatty acids such as isopropyl myristate, cetyl palmitate andoctyldodecylmyristate (Wickenol 142); beeswax; saturated and unsaturatedfatty alcohols such as behenyl alcohol and cetyl alcohol; fatty sorbitanesters; lanolin and lanolin derivatives; C.sub.1-C.sub.24 esters ofdimer and trimer acids such as diisopropyl dimerate, diisostearylmalate,diisostearyldimerate and triisostearyltrimerate; and silicones such aswater-insoluble silicones inclusive of non-volatile polyalkyl andpolyaryl siloxane gums and fluids, volatile cyclic and linearpolyalkylsiloxanes, polyalkoxylated silicones, amino and quaternaryammonium modified silicones, rigid cross-linked and reinforced siliconesand mixtures thereof.

It is possible to use standard antioxidants such as t-butylhydroquinone, butylated hydroxytoluene and .alpha.-tocopherol and itsderivatives, alpha lipoic acid, resveratrol and its derivatives in thecosmetic compositions of the present invention, preferably, in amountsless than would normally be utilized.

Similarly, it is possible to use standard preservatives such as methyl,ethyl, propyl, butyl and isobutyl p-hydroxybenzoate (parabens),2-phenoxyethanol, sorbic acid, potassium sorbate, hexamidinediisothionate, imidazolidinylurea (Germall 115) or preservativesmarketed under the names Kathon and Tridssan.

A wide variety of optional ingredients such as non-occlusivemoisturizers, humectants, gelling agents, neutralizing agents, perfumes,coloring agents and surfactants can be added to the presentlycontemplated cosmetic compositions.

A humectant may be present in an amount of from about 0.1% to about 20%,preferably from about 1% to about 10% and especially from about 2% toabout 5% by weight of the total composition. Suitable humectants includesorbitol, propylene glycol, butylene glycol, hexylene glycol,ethoxylated glucose derivatives, hexanetriol, glycerin, water-solublepolyglycerylmethacrylate lubricants (e.g., compositions available underthe trademark Lubrajel) and panthenols (e.g. D-panthenol).

A hydrophilic gelling agent may be present in an amount of from about0.01% to about 10%, preferably from about 0.02% to about 2% andespecially from about 0.02% to about 0.5% by weight of the totalcomposition. Suitable hydrophilic gelling agents include celluloseethers (e.g., hydroxyethyl cellulose, hydroxypropylmethyl cellulose),polyvinylalcohol, guar gum, hydroxypropyl guar gum and xantham gum, aswell as the acrylic acid/ethyl acrylate copolymers and the carboxyvinylpolymers sold under the trademark Carbopol.

Neutralizing agents, suitable for use in neutralizing acidic groupcontaining hydrophilic gelling agents, include sodium hydroxide,potassium hydroxide, ammonium hydroxide, monoethanolamine,diethanolamine and triethanolamine.

Other optional materials include keratolytic agents such as salicylicacid; proteins and polypeptides and derivatives thereof; soluble orcolloidally-soluble moisturizing agents such as hylaronic acid andstarch-grafted sodium polyacrylates; coloring agents; perfumes andperfume solubilizers; surfactants/emulsifiers such as fatty alcoholethoxylates and ethoxylated polyol fatty acid esters; and pigments whichcan be organic or inorganic and which include materials having a lowcolor or lustre, such as matte finishing agents, and also lightscattering agents.

Incorporation of a UV protective agent into the preparation of thepresent invention makes it possible to suppress undesired actions of UVlight that would otherwise undermine microcirculation and dermalfibroblast growth.

The UV screening agent which can be used in the present invention is notparticularly limited as long as it can be incorporated physicochemicallyinto the preparation of the present invention and is capable ofproducing the above-described synergistic effects when incorporated intothe preparation. Examples of suitable UV protective agents are shownbelow for illustrative purposes only, and should not be construed aslimiting.

UV-A absorbers include anthranilic acid derivatives, such as methylanthranilate and homomentyl N-acetylanthranilate; benzophenonederivatives, such as 2,4-dihydroxybenzophenone,2,2′-dihydroxy-4-methoxybenzophenone,2,2′-dihydroxy-4,4′-dimethoxybenzophenone,2,2′,4,4′-tetrahydroxybenzophenone, 2-hydroxy-4-methoxybenzophenone,2-hydroxy-4-methoxy-4′-methylbenzophenone,2-hydroxy-4-methoxybenzophenone-5-sulfonic acid salts,4-phenylbenzophenone, 2-ethylhexyl-4′-phenylbenzophenone-2-carboxylates,2-hydroxy-4-n-octoxybenzophenone, and 4-hydroxy-3-carboxybenzophenone;benzotriazole derivatives, such as2,2′-hydroxy-5-methylphenylbenzotriazole,2-(2′-hydroxy-5′-t-octylphenyl)benzotriazole and2-(2′-hydroxy-5′-methylphenyl)benzotriazole; dianisoylmethane, and4-methoxy-4′-t-butyldibenzoylmethane (Parsol A.TM.). Preferred of theseUV-A absorbers are 4-methoxy-4′-t-butyidibenzoylmethane,2-hydroxy-4-methoxybenzophenone, and 2-hydroxy-4-methoxybenzophenonederivatives, e.g., 2-hydroxy-4-methoxybenzophenone-5-sulfonic acidsalts, for their safety and efficacy.

Medium-wavelength ultraviolet (hereinafter referred to as UV-B)absorbers include benzoic acid derivatives, such as p-aminobenzoic acid(hereinafter abbreviated as PABA), glycerol mono-PABA ester,N,N-dipropoxyPABA ethyl ester, N, N-diethoxyPABA ethyl ester,N,N-dimethylPABA ethyl ester, N, N-dimethylPABA butyl ester, andN,N-dimethylPABA amyl ester; salicylic acid derivatives, such asdipropylene glycol salicylate, ethylene glycol salicylate, myristylsalicylate, methyl salicylate, amyl salicylate, mentyl salicylate,homomentyl salicylate, octyl salicylate, phenyl salicylate, benzylsalicylate, and p-isopropylphenyl salicylate; cinnamic acid derivatives,such as octyl cinnamate, ethyl 4-isopropylcinnamate, methyl2,5-diisopropylcinnamate, ethyl 2,4-diisopropylcinnamate, methyl2,4-diisopropylcinnamate, propyl p-methoxycinnamate, isopropylp-methoxycinnamate, isoamyl p-methoxycinnamate, octyl p-methoxycinnamate(2-ethylhexyl p-methoxycinnamate), 2-ethoxyethyl p-methoxycinnamate,cyclohexyl p-methoxycinnamate, ethyl.alpha.-cyano-.beta.-phenylcinnamate, 2-ethylhexyl.alpha.-cyano-.beta.-phenylcinnamate, glycerolmono-2-ethylhexanoyl-di-p-methoxycinnamate, octyl methoxycinnamate,3-methyl-4-ethylbis(trimethylsiloxy)silyl !butyl3,4,5-trimethoxycinnamate, and monoethyl p-dimethoxycinnamate; camphorderivatives, such as 3-(4′-methylbenzylidene)-d, 1-camphor,3-benzylidene-d, 1-camphor, and5-(3,3-dimethyl-2-norbornylidene)-3-pentene-2-one; urocanic acid, ethylurocanate, 2-phenyl-5-methylbenzoxazole, and dibenzalazine.

The UV screening agents include titanium oxide (TiO.sub.2), talc(MgSiO.sub.2), Carmine (FeO.sub.2), bentonite, kaolin, and zinc oxide(ZnO).

These UV protective agents can be incorporated into the preparation ofthe present invention in an appropriate combination thereof in agreementwith the particular purpose and form of the preparation. Collagencross-linking is a phenomenon which occurs mainly in the dermal layer ofthe skin. Where a UV protective agent is used for the specific purposeof affecting synergistic inhibitory action on collagen cross-linking, itis recommended to select UV-A absorbers which absorb UV-A (wavelength:320 to 400 nm) capable of reaching the dermal layer rather than UV-Babsorbers which absorb UV-B (wavelength: 280 to 320 nm). It isparticularly preferred in the present invention to positively use UV-Aabsorbers for the purpose of producing synergistic inhibitory effects oncollagen cross-linking.

The denotation “UV-A absorbers” or “UV-B absorbers” as used herein doesnot always mean that the agents are capable of absorbing only UV-A orUV-B but means that they are capable of absorbing at least UV-A or UV-B.For example, the benzophenone UV absorbers mentioned above as examplesof UV-A absorbers are capable of absorbing UV-B as well as UV-A.

The amount of the UV protective agent to be incorporated is subject tovariation depending on the properties to be imparted to the preparationof the present invention. It is usually 0.01 to 30% by weight,preferably 0.1 to 20% by weight, based on the total preparation. If theamount is less than 0.01% by weight, the agent tends to fail to bringabout synergistic effects that would have been obtained by addition of asufficient amount of the agent. If the amount exceeds 30% by weight, anyfurther enhancement of synergistic effects that might be expected fromemploying the increased amount hardly results.

The inclusion of a whitening effect in the preparation is effective foralleviating the bad influences of UV light on the skin. To this effect,a whitening agent, such as placental extract, glutathione, extract ofcreeping saxifrage (Saxifrage stolonifera), etc. may be added to thepreparation.

The provision of an anti-inflammatory effect in the preparation iseffective for alleviating the bad influences of UV light on the skin. Tothis effect, an anti-inflammatory agent, such as a glycyrrhizic acidderivative, a glycyrrhetic acid derivative, a salicylic acid derivative,hinokitiol, zinc oxide, etc. may be added to the preparation.

Over-the-Counter/Food and Drug Administration-approved skin soothingagents, such as, but not limited to dimethicone and allantoin can beadded to the preparation of the invention for further skin benefit.

It is also possible to add various plant extracts into the preparationfor various purposes. Examples of usable plant extracts arephellodendron extract, coptis roots extract, Lithospermi radix extract,Paeoniae radix extract, Lycium barbarum extract, Swertia extract, birchextract, Rhodiola extract, olive leaf extract, grapeseed extract, Arnicaextract, sage extract, loquart extract, ginseng extract, aloe extract,common mallow extract, iris extract, grape extract, Coicis semen (Coixlachryma-jobi) extract (Yokuinin), loofah (Luffa cylindrica Rosem)extract, Lilium extract, crocus extract, Cnidii rhizoma extract, gingerextract, Saint-John's wort (Hypericum erectum) extract, petty white(Ononis spinosa) root extract, rosemary extract, garlic extract,capsicum extract, and orange peel.

Vitamins may also be added to the preparation in order to impart variouspeculiar effects to the preparation, for example, a cutaneous aginginhibitory effect. Examples of usable vitamins are vitamin A's, such asvitamin A oil, retinol, retinol acetate; vitamin B.sub.1; vitaminB.sub.2's, such as riboflavin, riboflavin butyrate, and flavin adeninedinucleotide; B.sub.3; vitamin B.sub.5; vitamin B.sub.6 ′s, such aspyridoxine hydrochloride and pyridoxine dioctanoate; vitamin B.sub. 12;vitamin C's, such as L-ascorbic acid, L-ascorbic acid palmitate,L-ascorbic acid 2-sulfate, L-ascorbic acid phosphate, andDL-.alpha.-tocopherol-L-ascorbic acid phosphate diester dipotassium;pantothenic acid derivatives, such as calcium pantothenate,D-pantothenyl alcohol, pantothenyl ethyl ether, and acetylpentothenylethyl ether; vitamin D's, such as ergocalciferol and cholecalciferol;nicotinic acid compounds, such as nicotinic acid, nicotinamide, andbenzyl nicotinate; vitamin E's, such as .alpha.-tocopherol, tocopherolacetate, DL-.alpha.-tocopherol nicotinate, and DL-.alpha.-tocopherolsuccinate; and other vitamins such as vitamin P; folate and biotin.

Minerals may also be added to the preparation in order to impartmicroelements required for the optimal skin metabolism, such asmagnesium, zinc and copper, under various forms such as, but not limitedto magnesium aspartate, zinc gluconate or copper gluconate.

Other active ingredients, which may be incorporated into the preparationof the present invention include peptides, amino acids, polysaccharidesand peptidoglycans.

The active ingredients which may be incorporated into the preparation ofthe present invention are not limited to the above-enumerated specificexamples. Further, the pharmacological efficacy of the above-enumeratedactive ingredients is not limited to the one described. For example,vitamin C's are useful as not only a whitening agent but also anantioxidation assistant. These active ingredients may be incorporatedinto the preparation cither individually or as an appropriatecombination of two or more thereof according to the purpose prescribedfor the preparation.

The present invention is broadly applicable to cosmetics,pharmaceuticals and non-medical applications which are externallyapplied to the skin and may take a wide variety of forms, such asaqueous solutions, solubilized systems, emulsions, powders, oilyliquids, gels, ointments, aerosols, water-oil two phase systems,water-oil-powder three phase systems, and the like. For example, thepreparations of the present invention are-applicable to a wide range ofcosmetic forms, such as facial cleansers, clear lotions, milky lotions,creams, jellies, skin revitalizers, and facial packs as skin carecosmetics, and foundation creams as makeup cosmetics; and a wide rangeof pharmaceutical or non-medical application forms, such as ointments.The forms applicable to the preparations of the present invention are byno means limited to the above-mentioned specific dose forms.

The preparations according to the present invention may comprise a widevariety of known bases or vehicles according to the desired dose form aslong as the effects of the present invention are not impaired. Suchbases or vehicles include oils, e.g., avocado oil, tubaki oil (camelliaoil), evening primrose oil, Turtle oil, Macadamia nut oil, corn oil,mink oil, olive oil, rape oil, egg yolk oil, sesame oil, persic oil(apricot kernel oil), wheat germ oil, sasanqua oil, castor oil, linseedoil, safflower oil, cotton seed oil, perilla oil, soybean oil, peanutoil, tea seed oil, kaya (Torreya nucifera) oil, rice bran oil, Chinesetung oil, Japanese tung oil, jojoba oil, germ oil, triglycerides (e.g.,glycerol trioctanoate and glycerol triisopalmitate); fats, such as cacaofat, coconut oil, horse fat, hardened coconut oil, palm oil, beeftallow, mutton tallow, hardened beef tallow, palm kernel oil, lard, beefbone fat, haze (Rhus succedancea L.) kernel oil, hardened oil, beef footoil, Japan wax, and hardened castor oil; waxes, such as molasses,candelilla wax, cotton wax, carnauba wax, bayberry wax, cera ibota,whale wax, montan wax, rice bran wax, lanolin, kapok wax, lanolinacetate, liquid lanolin, sugar cane wax, lanolin fatty acid isopropylester, hexyl laurate, hydrogenated lanolin, jojoba wax, hard lanolin,Shellac wax, polyoxyethylene (hereinafter abbreviated as POE) lanolinalcohol ether, POE lanolin alcohol acetate, POE cholesterol ether,polyethylene glycol lanolin fatty acid ester, and POE hydrogenatedlanolin alcohol ether; hydrocarbon oils, such as liquid paraffin,ozokerite, squalene, pristane, paraffin, ceresin, vaseline, andmicrocrystalline wax; higher fatty acids, e.g., lauric acid, myristicacid, palmitic acid, stearic acid, behenic acid, oleic acid,12-hydroxystearic acid, undecylenic acid, Tall oil, isostearic acid,linoleic acid, linolenic acid, eicosapentaenoic acid (EPA), anddocosahexaenoic acid (DHA); higher alcohols including straight-chainalcohols, such as lauryl alcohol, cetyl alcohol, stearyl alcohol,behenyl alcohol, myristyl alcohol, oleyl alcohol, and cetostearylalcohol, and branched alcohols, such as monostearyl glycerol ether(batyl alcohol), 2-decyltetradecinol, lanolin alcohol, cholesterol,phytosterol, hexyldodecanol, isostearyl alcohol, and octyidodecanol;synthetic ester oils, such as isopropyl myristate, cetyl octanoate,octyidodecyl myristate, isopropyl palmitate, butyl stearate, hexyllaurate, myristyl myristate, decyl oleate, hexyldecyl dimethyloctanoate,cetyl lactate, myristyl lactate, lanolin acetate, isocetyl stearate,isocetyl isostearate, cholesteryl 12-hydroxystearate, ethylene glycoldi-2-ethylhexylate, dipentaerythritol fatty acid ester, N-alkyl glycolmonoisostearate, neopentyl glycol dicaprate, diisostearyl malate,glycerol di-2-heptylundecanoate, trimethylolpropane tri-2-ethylhexylate,trimethylolpropane triisostearate, pentaerythritoltetra-2-ethylhexylate, glycerol tri-2-ethylhexylate, trimethylolpropanetriisostearate, cetyl 2-ethylhexylate, 2-ethylhexyl palmitate, glyceroltrimyristate, glycerol tri-2-heptylundecanoate, castor oil fatty acidmethyl ester, oleic acid oil, cetearyl alcohol, acetglyceride,2-heptylundecyl palmitate, diisobutyl adipate, 2-octyidodecylN-lauroyl-L-glutamate, di-2-heptylundecyl adipate, ethyl laurate,di-2-ethylhexyl sebacate, 2-hexyldecyl myristate, 2-hexyldecylpalmitate, 2-hexyldecyl adipate, diisopropyl sebacate, 2-ethylhexylsuccinate, ethyl acetate, butyl acetate, amyl acetate, and triethylcitrate; silicone derivatives including chain polysiloxanes, such asdimethyl polysiloxane, methylphenyl polysiloxane, and methylhydrogenpolysiloxane, cyclic polysiloxanes, such as decamethyl polysiloxane,dodecamethyl polysiloxane, and tetramethyltetrahydrogen polysiloxane,silicone resins forming a three-dimensional network structure, andsilicone rubber; anionic surface active agents, such as soap base, fattyacid soaps (e.g., sodium laurate or sodium palmitate), higheralkylsulfates (e.g., sodium lauryl sulfate and potassium laurylsulfate), alkyl ether sulfates (e.g., POE triethanolamine lauryl sulfateand POE sodium lauryl sulfate), N-acylsarcosine (e.g., sodiumlauroylsarcosine), higher fatty acid amide sulfonates (e.g., sodiumN-myristoyl-N-methyltaurine, sodium coconut-oil fattyacid-methyltaurine, and sodium laurylmethyltaurin), phosphoric estersalts (e.g., sodium POE oleyl ether phosphate and POE stearyl etherphosphoric acid), sulfosuccinates (e.g., sodiumdi-2-ethylhexylsulfosuccinate, sodium monolauroylmonoethanolamidepolyoxyethylene sulfosuccinate, and sodium lauryl polypropylene glycolsulfosuccinate), alkylbenzenesulfonates (e.g., sodium lineardodecylbenzenesulfonate, triethanolamine linear dodecylbenzenesulfonate,linear dodecylbenzenesulfonic acid), N-acylglutamates (e.g., sodiumN-lauroylglutamate, disodium N-stearoylglutamate, and monosodiumN-myristoyl-L-glutamate), higher fatty acid ester sulfates (e.g., sodiumglycerol hardened coconut oil fatty acid sulfate), sulfated oils (e.g.,Turkey red oil), POE alkyl ether carboxylic acids, POE alkyl allyl ethercarboxylates, .alpha.-olefinsulfonates, higher fatty acid estersulfonates, secondary alcohol sulfates, higher fatty acidalkylolamidosulfates, sodium lauroyl monoethanolamidosuccinate,ditriethanolamine N-palmitoylaspartate, and casein sodium; cationicsurface active agents, such as alkyltrimethylammonium salts (e.g.,stearyltrimethylammonium chloride and lauryltrimethylammonium chloride),distearyldimethylammonium chloride, alkylpyridinium salts (e.g.,poly(N,N′-dimethyl-3,5-methylenepiperidinium) chloride andcetylpyridinium chloride), alkyl quaternary ammonium salts,alkyldimethylbenzylammonium salts, alkylisoquinolinium salts,dialkylmorpholinium salts, POE alkylamines, alkylamine salts, polyaminefatty acid derivatives, amyl alcohol fatty acid derivatives,benzalkonium chloride, and benzethonium chloride; amphoteric surfaceactive agents, such as imidazoline type amphoteric surface active agents(e.g., sodium 2-undecyl-N,N,N-(hydroxyethylcarboxymethyl)-2-imidazolineand 2-cocoyl-2-imidazolinium hydroxide-1-carboxyethyloxy disodium), andbetaine type amphoteric surface active agents (e.g.,2-heptadecyl-N-carboxymethyl-N-hydroxyethylimidazolinium betaine,lauryldimethylaminoacetic acid betaine, alkylbetaines, amidobetaines,and sulfobetaines; lipophilic nonionic surface active agents, such assorbitan fatty acid esters (e.g., sorbitan mono-oleate, sorbitanmonoisostearate, sorbitan monolaurate, sorbitan monopalmitate, sorbitanmonostearate, sorbitan sesquioleate, sorbitan trioleate, diglycerolsorbitan penta-2-ethylhexylate, and diglycerol sorbitantetra-2-ethylhexylate), glycerol fatty acid esters (e.g., glycerolmono-cotton seed oil fatty acid ester, glycerol monoerucate, glycerolsesquioleate, glycerol monostearate, glycerol .alpha., .alpha.′-oleatepyroglutamate, and glycerol monostearate malate), propylene glycol fattyacid esters (e.g., propylene glycol monostearate), hardened castor oilderivatives, glycerol alkyl ethers, and polyoxyethylene-methylpolysiloxane copolymers; hydrophilic nonionic surface active agents,such as POE sorbitan fatty acid esters (e.g., POE sorbitan mono-oleate,POE sorbitan monostearate and POE sorbitan tetraoleate), POE sorbitolfatty acid esters (e.g., POE sorbitol monolaurate, POE sorbitolmono-oleate, POE sorbitol pentaoleate, and POE sorbitol monostearate),POE glycerol fatty acid esters (e.g., POE glycerol monostearate, POEglycerol monoisostearate, and POE glycerol triisostearate), POE fattyacid esters (e.g., POE mono-oleate, POE distearate, POE monodioleate,and ethylene glycol distearate), POE alkyl ethers (e.g., POE laurylether, POE oleyl ether, POE stearyl ether, POE behenyl ether, POE2-octyldodecyl ether, and POE cholestanol ether), POE alkyl phenylethers (e.g., POE octyl phenyl ether, POE nonyl phenyl ether, and POEdinonyl phenyl ether), polyoxyethylene polypropylene glycol ether (e g.,Pluronic), POE-POP alkyl ethers (e.g., POE-POP cetyl ether, POE-POP2-decyltetradecyl ether, POE-POP monobutyl ether, POE-POP hydrogenatedlanolin, and POE-POP glycerol ether), tetra POE-tetra POPethylenediamine condensates (e.g., Tetronic), POE castor oil or hardenedcastor oil derivatives (e.g., POE castor oil, POE hardened castor oil,POE hardened castor oil monoisostearate, POE hardened castor oiltriisostearate, POE hardened-castor oil monopyroglutamatemonoisostearate, and POE hardened castor oil maleate), POE molasseslanolin derivatives (e.g., POE sorbitol molasses), alkanolamides (e.g.,coconut oil fatty acid diethanolamide, lauric monoethanolamide, andfatty acid isopropanolamide), POE propylene glycol fatty acid esters,POE alkylamines, POE fatty acid amides, sucrose fatty acid esters, POEnonylphenyl formamide condensate, alkylethoxydimethylamine oxides, andtrioleyl phosphate; antiseptics such as methyl p-hydroxybenzoate, ethylp-hydroxybenzoate, and butyl p-hydroxybenzoate; masking agents, such asdisodium edetate and EDTA; naturally occurring water-soluble highpolymers including vegetable high polymers, such as gum arabic,tragacanth, galactan, guar gum, carob gum, karaya gum, carrageenan,pectin, agar, quince seeds, algae colloid (brown algae extract), starch(rice, corn, potato or wheat), and glycyrrhizic acid, microorganism highpolymers, such as xantham gum, dextran, succinoglucan, and pullulan, andanimal high polymers, such as collagen, casein, albumin, and gelatin;semisynthetic water-soluble high polymers, such as starch high polymers(e.g., carboxymethyl starch and methylhydroxypropyl starch), cellulosehigh polymers (e.g., methyl cellulose, nitrocellulose, ethyl cellulose,methylhydroxypropyl cellulose, hydroxyethyl cellulose, sodium cellulosesulfate, hydroxypropyl cellulose, carboxymethyl cellulose (CMC) sodium,microcrystalline cellulose, and powdered cellulose), and alginic acidhigh polymers (e.g., sodium alginate and propylene glycol alginate);synthetic water-soluble high polymers, such as vinyl polymers (e.g.,polyvinyl alcohol, polyvinyl methyl ether, polyvinylpyrrolidone,carboxyvinyl polymer (Carbopol), and alkyl-modified carboxyvinylpolymers), polyoxyethylene high polymers (e.g., polyethylene glycol2000, 4000 or 6000), polyoxyethylene-polyoxypropylene copolymers,acrylic polymers (e.g., sodium polyacrylate, polyethyl acrylate, andpolyacrylamide), polyethylene-imine, and cationic polymers; inorganicwater-soluble high polymers, such as bentonite, magnesium aluminumsilicate, laponite, hectorite, and silicic anhydride; thickeners, suchas gum arabic, carrageenan, karaya gum, tragacanth, carob gum, quinceseeds, casein, dextrin, gelatin, sodium pectate, sodium alginate, methylcellulose, ethyl cellulose, CMC, hydroxyethyl cellulose, hydroxypropylcellulose, PVA, PVM, PVP, sodium polyacrylate, carboxyvinyl polymer,locust beam gum, guar gum, tamarind gum, cellulosedialkyldimethlyammonium, sulfate, xantham gum, aluminum magnesiumsilicate, bentonite, and hectorite; powder components includinginorganic powders, such as talc, kaolin, mica, sericite, commonmica,phlogopite, synthetic mica, lepidolite, biotite, lithia mica,vermiculite, magnesium carbonate, calcium carbonate, aluminum silicate,barium silicate, calcium silicate, magnesium silicate, strontiumsilicate, metal tungstates, magnesium, silica, zeolite, barium sulfate,calcined calcium sulfate (calcined gypsum), calcium phosphate,fluorapatite, hydroxyapatite, ceramic powder, metal soaps (e.g., zincmyristate, calcium palmitate and aluminum stearate), and boron nitride,and organic powders, such as polyamide resin powder (nylon powder),polyethylene powder, polymethyl methacrylate powder, polystyrene powder,styrene-acrylic acid copolymer resin powder, benzoguanamine resinpowder, polytetrafluoroethylene powder, and cellulose powder; colorants,such as inorganic white pigments (e.g., titanium dioxide and zincoxide), inorganic red pigments (e.g., iron oxide and iron titanate),inorganic brown pigments (e.g., .gamma.-iron oxide), inorganic yellowpigments (e.g., yellow iron oxide and ocher), inorganic black pigments(e.g., black iron oxide, carbon black, and titanium oxide of low order),inorganic purple pigments (e.g., mango violet and cobalt violet),inorganic green pigments (e.g., chromium oxide, chromium hydroxide, andcobalt titanate), inorganic blue pigments (e.g., ultramarine andPrussian blue), pearlescent pigments (e.g., titanium oxide-coated mica,titanium oxide-coated bismuth oxychloride, titanium oxide-coated talc,colored titanium oxide-coated mica, bismuth oxychloride, and fish scaleguanine), metal powder pigments (e.g., aluminum powder and copperpowder), organic pigments (e.g., Red No. 201, Red No. 202, Red No. 204,Red No. 205, Red No. 220, Red No. 226, Red No. 228, Red No. 405, OrangeNo. 203, Orange No. 204, Yellow No. 205, Yellow No. 401, and Blue No.404), zirconium, barium or aluminum lake organic pigments (e.g., Red.No. 3, Red No. 104, Red No. 106, Red No. 227, Red No. 230, Red No. 401,Red No. 505, Orange No. 205, Yellow No. 4, Yellow No. 5, Yellow No. 202,Yellow No. 203, Green No. 3, and Blue No. 1), natural dyes (e.g.,chlorophyll and beta.-carotene), Titan Yellow, carthamin, and safflower;perfumes; water; alcohols; and the like.

The compositions of the present invention may be prepared by anyconventional technique for preparing a cosmetic composition by merelysubstituting an aqueous extract from Angelica sinensis or a fraction ora lyophilizate thereof, or one or more active components contained insaid extract for the water normally incorporated into the composition.

The present invention having been described in detail in the precedingsections, the following examples are provided to illustrate certainaspects of, but not to limit, the invention.

EXAMPLES Example 1

Stimulatory Effect of SBD.4 Purified by DEAE on the Proliferation ofEndothelial Cells.

This example illustrates the SBD.4 stimulation of bovine capillaryendothelial (BCE) cell growth in a dose-dependant manner, in vitro. FIG.1 demonstrates this effect with SBD.4 angiostimulatory extract purifiedby the means of filtration and DEAE-cellulose. Briefly, dried,fragmented roots of Angelica sinensis were placed in water (w:v=1:20),brought to boil and boiled for 30 minutes at 95 degrees centigrade. Theaqueous extract was then cooled to room temperature, centrifuged toremove insoluble solids and sterile-filtered through a 0.22 micronNalgene filter. This filtrate was passed through a centrifugal filterdevice (Millipore) of 10,000 daltons molecular weight cut-off and theangiostimulatory material was collected in the lower chamber.Thirty-five milliliters of this angiostimulatory material was thenapplied to 8 centimeters×1 centimeter DEAE column.

The angiostimulatory activity was eluted by the aqueous solution of fivehundred millimolar (mM) sodium chloride with one hundred millimolarhydrochloric acid. The flow rate through the column during the wholeprocess was 2 milliliter/minute.

The angiostimulatory material eluted from the DEAE column was tested onthe proliferation of bovine capillary endothelial cells in 96 welltissue culture plate. Cells were plated at about 3,000 cells per well inDMEM medium (Hyclone, South Logan, Utah) supplemented by 5% of bovinecalf serum (Hyclone, South Logan, Utah). The following day threedifferent concentrations of SBD.4 eluted from the DEAE column were addedto cells (final concentrations: 0.5 microgram per milliliter (μg/ml), 2μg/ml and 10 μg/ml). One nanogram per milliliter (ng/ml; finalconcentration) of bFGF was added to other wells as control. Cells werecounted seventy-two hours later. Cell number was estimated using thesulforhodamine B (Sigma, St. Louis, Mo.) colorimetric assay (Skehan etal., 1990) The results were graphically represented, with the number ofcells represented as optical density (OD) readings at 570 nanometerswavelength of sulforhodamine B staining, plotted on the Y axis (see FIG.1). The extent of this stimulation is comparable with bFGF, which is amajor angiostimulatory factor in humans. The mechanism of action ofSBD.4 may involve the stimulation of the bFGF and/or VEGF productionand/or binding to endothelial cells and/or interference with the nitricoxide pathways.

Example 2

SBD.4 Stimulates Proliferation of Capillary Endothelial Cells, andVessel-Like Structure Formation in a Tri-Dimensional Model by SBD.4.

As shown in the FIG. 2, HPLC-purified SBD.4 strongly stimulates anotherbatch of capillary endothelial cells (compare “Control” and “SBD.4”bars). Furthermore, this figure shows that SBD.4 is significantly morestable at room temperature than basic fibroblast growth factor (bFGF),and unlike bFGF, it is pronase-insensitive (compare “SBD.4(RT)” with“bFGF(RT)” and “SBD.4/Pronase” with “bFGF/Pronase”.

It is known in the art that pronase is a potent combination of severalendo- and exo- proteinases, which can cleave almost any peptide bond.These results suggest that SBD.4 may maintain its activity in theproteolytic environment of the wound to much greater extent thanproteinaceous growth factors tested for wound healing, such as bFGF andPDGF (platelet derived growth factor).

As demonstrated in FIG. 3, SBD.4 (purified by DEAE and HPLC) is alsocapable of inducing capillary-like structures in a tridimensional tubeformation model developed in our laboratory. In this model,capillary-like structures are induced by plating bovine endothelialcells (about 8000 cells per well) in a 96 well tissue culture-treatedplate (Costar) in DMEM supplemented with 5% calf serum (Hyclone, Logan,Utah), with or without an endothelial cell stimulatory growth factor.Cells cultured for 2-5 weeks with such a stimulatory growth factor (forexample bFGF, FIG. 3C) form capillary-like structures. Cells grownwithout such factor form a monolayer (FIG. 3A).

This example shows that SBD.4 (5 μg/ml; FIG. 3B) induces a remodelingresponse, leading to the generation of a tridimensional network ofvessel-like structures, similar to, and often more complete than the oneinduced by the optimal concentration of bFGF (15 ng/ml, ornanograms/milliliter; FIG. 3C), used as positive control.

Example 3

SBD.4 Stimulates Angiogenesis In Vivo, in the Chorioallantoic Membrane(CAM) Assay:

The chorioallantoic membrane (CAM) is a highly vascularized structurepresent in fertilized, developing hen egg. The sensitive,semi-quantitative assay using CAM has been used for testing manyangiogenesis inhibitors and stimulators (Ribatti et al., 2000). Theassay consists of placing a methyl-cellulose pellet containing thecompound of interest on the CAM of an 8 day-old chick fertilized egggrown in a Petri dish. The compound (in our case SBD.4) is graduallyreleased from the pellet and its effect on the embryo vasculature(induction of new blood vessels) is assessed optically. FIG. 4illustrates the result of 3kD cut off filtration-purified SBD.4 on thevascularization in this assay. When a methylcellulose pellet with SBD.4is placed on the CAM (in the center of the panel A; it is hardlyvisible, because it is transparent), one can observe generation ofseveral new blood vessel growing towards the pellet as early as 24 hours(24 h) later (note for example a hook-like red capillary sprouting fromthe preexisting vessel in the center of the panel B). Panel Cillustrates a typical “spokes of a wheel” pattern of new blood vesselsconverging towards the pellet with Angelica (darker area in the centerof the panel C) 48 hours (48 h) after the beginning of a differentexperiment. Together, these experiments demonstrate the angiostimulatorypotential of SBD.4 in this in vivo system.

Example 4

Stimulatory Effect of SBD.4 on Human Dermal Fibroblasts

This example shows that DEAE- and HPLC-purified SBD.4 has a stimulatoryeffect on human dermal fibroblasts and on type I collagen output. Humanneonatal dermal fibroblasts (passage 3-5; Cambrex, Walkersville, Md.)were cultured at 5% CO₂ in complete fibroblast growth medium (FGM-2bulletkit; Cambrex cat.# CC-3132). After 3 days this medium wasreplaced. 0.05% glucose (negative control) or SBD.4 was added to cells,which were then incubated one week. At the end of the experiment, cellnumber was determined by a colorimetric method, according to Skehan andcoll. (1990). We found that nanogram amounts of SBD.4 stimulated humandermal fibroblast growth (FIG. 5A) and collagen I concentration infibroblast-conditioned medium (FIG. 5B). Longer periods of incubation atsuboptimal conditions (for example 1 month) resulted in even biggerdifferences in cell number and collagen output between controls andSBD.4- treated cells, in favor of the latter.

Example 5

SBD.4 Enhances Wound Closure in Diabetic Mice.

We have chosen to use the genetically diabetic mice (13 week old femaleBKS Cg-m +/+ Lepr(db), stock # 642, Jackson Labs) because it is anoptimal model reflecting the impaired wound healing in a segment of thepopulation particularly vulnerable to chronic wounds—the diabeticpatients. This genetically diabetic mouse model is well established asdocumented by over 50 publications (for example see Tsuboi et al.,1992). Many companies have also used it as a standard pre-clinicalmodel, including Johnson & Johnson, which included it in thepre-clinical data package submitted to FDA as part of their INDapplication for becaplermin.

Two concentrations of saline solutions of SBD.4 (3 kd cut-off filteredaqueous extract): 10 mg per wound and 2 mg per wound (20 mg/cm², and 4mg/cm², respectively) against saline (PBS) as negative control. Theconcentrations of SBD.4 have been based on the fact that there is a 3 to4 log difference between the concentration of bFGF required for thestimulation of endothelial cells in vitro and the bFGF concentrationactive in diabetic mouse wounds (Greenhalgh et al., 1990). Thus, we alsochose this multiplication factor for our angiostimulator.

Twelve mice were divided in 2 groups and 8 mm (diameter) full thicknessexcisional wounds were performed under anesthesia on each side of theanimal with a punch biopsy tool and covered by sterile gauze (occlusivedressings were not used as they considerably delay wound healing,according to the experience of the Principal Inventor and to thepublished reports (Lasa et al., 1993). The number of animals per groupwas based on the previous publications with this model; Jacobi et al.,2002). In Group 1 (the experimental treatment group), one wound on eachanimal was treated by 50 ul of “high” concentration of SBD.4 (10 mg perwound) and the other one by 50 ul of “low” concentration of SBD.4 (2 mgper wound). In Group 2 (negative control), both wounds were treated byPBS (phosphate buffered saline). Treatments were applied on the firstday and every second day thereafter. Wound surface were measured everysecond day. Wound closure was determined by the following formula:percent closure={(area on day 0−open area)/area on day 0}×100. Woundswere considered closed if moist granulation tissue was no longerapparent, and the wound appeared covered with new epithelium.

All mice survived until the end of the experiment. However, the woundsof one control mouse refused to heal through the course of theexperiment. This was in contrast with other control mice, whose woundsslowly progressed towards healing. Therefore, the control mouse withnon-healing wounds has been excluded from the computational evaluation.As shown in Table 1, both concentrations of SBD.4 stimulated woundclosure, compared to the negative control. There was no significantadvantage in using 10 mg over 2 mg of the product per wound. Therefore,2 mg SBD.4 per wound has been chosen for further experiments. TABLE IMedian percentage of wound closure in genetically diabetic mice. Woundswere treated by 10 mg SBD.4 (High SBD.4); 2 mg SBD.4 (Low SBD.4) or byPBS (control). The overall stimulation by low and high concentrations ofSBD.4 was comparable, and thus the lower dose was chosen for furtherstudies. The results were expressed as medians, as this representationis more statistically significant for small data sets (6 mice treatedwith SBD.4 and 5 controls - one control mouse has been removed from theset due to non-healing wounds). Low High Stimulation by Stimulation byControl SBD.4 SBD.4 Low SBD.4 High SBD.4 (% (% (% relative to relativeto wound wound wound Control Control Day closure) closure) closure)(control = 1) (control = 1) 1 0 0 0 — — 3 30 32.11 34.6 1.07 1.153 535.3 48 43.3 1.36 1.227 6 38.8 51 54.9 1.314 1.415 8 51.2 73.4 68.81.434 1.344 10 75 88 89.5 1.173 1.193

Example 6

SBD.4 Enhances Wound Closure Better Than Becaplermin In Diabetic Mice.

This example illustrates the wound-healing stimulatory activity of theoptimal (in the limits determined in Example 5) concentration of SBD.4.Also, in this experiment we compared the bioactivity of 3 kD cutofffilter-purified SBD.4 to becaplermin (Regranex, Johnson & Johnson; thepositive control).

Twelve mice were divided in two groups and two wounds per mouse wereeffectuated as in Example 5. In order to exclude the possibility ofmouse-to-mouse variability in wound healing, in this experiment everymouse was treated by the test sample on one wound and by PBS (negativecontrol) on another wound. Thus, one group of 6 mice was treated with 50ul of SBD.4 (2 mg/wound) on one wound, and by 50 ul of PBS on the otherwound, while the second group of 6 mice was treated with becaplermin(positive control; 5 ug in 50 ul CMC—carboxymethylcellulose—gel) on oneand PBS on another wound. The concentration of becaplermin was based onthe dose used by Johnson & Johnson in their pre-clinical studies (2) andon published studies with PDGF-B, in the db/db model (Greenhalgh et al.,1990).

Treatments were applied on the first day and every second daythereafter. Wound surface were measured every second day. Wound closurefor each wound was determined by the formula: percent closure={(area onday 0−open area)/area on day 0}×100. Wounds were considered closed ifmoist granulation tissue was no longer apparent, and the wound appearedcovered with new epithelium. The dynamics of wound closure for the 2groups is plotted on FIG. 6 (means) and Table II (medians). Bothrepresentations show a significant stimulation of wound closure by SBD.4as compared with the negative control (PBS). This stimulation issuperior to the effect achieved with becaplermin.

Example 7

SBD.4 Enhances Wound Closure in Human/SCID Mouse Chimera Model.

Materials and Methods

Human skin graft in severe combined immunodeficient (SCID) mice is apowerful model for evaluating the effect of wound-healingdrug-candidates on full-thickness wounds (Juhasz et al., 1993). Thewound model in human skin/SCID chimera features normal migration ofneutrophils and macrophages to the wound bed, as well as normalexpression of growth factors associated with wound-healing, andtherefore provides a good approximation of human dermal wound-healingprocess (Matsumoto et al., 1997). The disadvantage of this model is thatit is delicate to handle and time-consuming. The donor skin must beimmediately placed on ice and taken to the lab, where a 10-hour graftingprocedure follows. We used surgical waste skin obtained under informedconsent from a patient undergoing abdominoplasty. TABLE II Medianpercentage of wound closure. Wounds were treated by 2 mg SBD.4; 5 ugbecaplermin or by PBS (control). There was a strong, consistentstimulation of wound closure by SBD.4. The positive control(Becaplermin) also stimulated closure, however, the stimulation by SBD.4was significantly stronger. Controls: n = 12; SBD.4: n = 6, Becaplermin:n = 6. Stimulation Stimulation by by SBD.4 becaplermin Control SBD.4relative relative to (% (% Becaplermin to Control Control Day closure)closure) (% closure) (control = 1) (control = 1) 1 0 0 0.0 — — 3 1.613.0 1.7 8.1 1.1 5 28.5 47.6 11.9 1.7 0.4 7 36.3 58.1 41.2 1.6 1.1 951.5 89 65.8 1.7 1.3 11 66.0 97.9 86.7 1.5 1.3Skin was defatted (if it is not well defatted, the remaining adiposetissue easily undergoes necrosis, which interferes with graft taking.But on the other side, removing the underlying fat tissue too slowly ortoo zealously can also damage the skin and compromise the graft), andgrafted (2.5×2.5 cm) on 15 SCID mice (Charles River Labs), using 10-12stitches per graft. Here, the challenge is to suture the thick humanskin with the much thinner rodent tissue.After 6 weeks, when the graft anastomosed with the murine skin 8 mm(diameter) full thickness wounds were excised in the human skin. Micereceived treatment (ointment every two days) of either becaplermin(Regranex, positive control), 2% carboxymethylcellulose (CMC, negativecontrol) or SBD.4 in 2% CMC on the day of wounding and every second daythereafter. Initial wound closure (defined by the coverage of the baseof the wound with bridging granular tissue and new epithelium) wasassessed every 3 days.Results and Discussion:

As shown in Table III, there was a dramatic increase in the initialwound closure in mice treated by SBD.4. On day 6, in the control group,none of the wounds was closed. In contrast, in the SBD.4 group, allwounds were closed. Control wounds (PBS-treated) achieved the same ratioof closure only at day 16. TABLE III Initial wound closure (defined aspresence of bridging tissue, no moist granulation tissue,epithelialization) on day 6 in human grafts treated with PBS (negativecontrol), becaplermin (positive control) and SBD.4. On Day 6, all woundsin SBD.4-treated animals were closed, only one wound in the Regranex(becaplermin) group was closed and no vehicle (CMC) -treated wounds wereclosed. N = 5 for all groups until day 6, when 1 representative mousefrom each group was sacrificed. N = 4 for days 7-16. # animals showinginitial Group: wound closure on: Control (PBS) Becaplermin SBD.4 Day 30/5 0/5 0/5 Day 6 0/5 1/5 5/5 Day 9 1/4 2/4 4/4  Day 12 2(3?)/4     4/44/4  Day 16 4/4 4/4 4/4

Example 8

Composition of SBD.4 at Three Representative Levels of Purity

This example shows the composition of SBD.4 at successive stages ofpurification, as visualized by HPLC-MS (FIG. 7). The endothelial- andfibroblast-stimulatory activity of SBD.4 is retained as the number ofmolecular entities is reduced from multiple in the aqueous extractpurified by molecular weight cut-off filtration (FIG. 7A-B), to lessthan 10 in the same extract further purified by reverse phase HPLC (FIG.7C-D). This number is further reduced when HPLC is preceded byDEAE-cellulose chromatography followed by methanol precipitation (FIG.7E-F). The most far-right peak on panels 7C and 7D is an impurity comingfrom the column and is not part of SBD.4.

REFERENCES

Dobak J, Grzybowski J, Liu F T, Landon B, Dobke M (1994)1,25-Dihydroxyvitamin D3 increases collagen production in dermalfibroblasts. J Dermatol Sci 8:18

Greenhalgh D G, Sprugel K H, Murray M J, Ross R. PDGF and FGF stimulatewound healing in the genetically diabetic mouse. Am J Pathol. 1990 June;136(6):1235

Jacobi J, Jang J J, Sundram U, Dayoub H, Fajardo L F, Cooke J P.Nicotine accelerates angiogenesis and wound healing in geneticallydiabetic mice. Am J Pathol. 2002, 161:97

Juhasz I, Murphy G F, Yan H C, Herlyn M, Albelda S M. Regulation ofextracellular matrix proteins and integrin cell substratum adhesionreceptors on epithelium during cutaneous human wound-healing in vivo.Am. J. Pathol. 1993; 143:1458

Laham R J, Chronos N A, Pike M, Leimbach M E, Udelson J E, Pearlman J D,Pettigrew R I, Whitehouse M J, Yoshizawa C, Simons M. Intracoronarybasic fibroblast growth factor (FGF-2) in patients with severe ischemicheart disease: results of a phase I open-label dose escalation study. JAm Coll Cardiol. 2000 36:2132

Lasa C I Jr, Kidd R R 3rd, Nunez H A, Drohan W N. Effect of fibrin glueand opsite on open wounds in DB/DB mice. J Surg Res. 1993 54:202

Matsumoto K, Robb E, Warden G, Nordlund J. The expression of cytokines,growth factors and ICAM-1 in the healing of human cutaneous xenograftson nude mice. Exp. Dermatol. 1997; 6:13

Ribatti D, Vacca A, Roncali L, Dammacco F. The chick embryochorioallantoic membrane as a model for in vivo research onanti-angiogenesis. Curr Pharm Biotechnol. 2000 1:73

Skehan P, Storeng R, Scudiero D, Monks A, McMahon J, Vistica D, Warren JT, Bokesch H, Kenney S, Boyd M R. New colorimetric cytotoxicity assayfor anticancer-drug screening. J. Natl. Cancer Inst. 1990; 82:1107

Tsuboi R, Shi C M, Rifkin D B, Ogawa H. A wound healing model usinghealing-impaired diabetic mice. J Dermatol. 1992 19:673

1. A method of promoting angiogenesis in a mammalian tissue comprisingdirectly contacting said tissue with an effective amount of Angelicasinensis extract SBD.4 or a fraction or a lyophilizate thereof, or oneor more active components contained in said extract to promoteangiogenesis in said tissue.
 2. The method of claim 1, wherein saidtissue is selected from the group consisting of endothelial, cardiac,cerebral, muscular, vascular, bone, fat, transplanted, dermal, epidermaland wounded.
 3. The method of claim 1, wherein said tissue is anischemic tissue selected from the group consisting of myocardialischemic tissue, cerebral ischemic tissue, veno-occlusive diseasedtissue, wounded tissue, damaged dermal tissue, damaged epidermal tissue,diabetic tissue and myocardial ischemic tissue wherein said myocardialischemic tissue is implicated in coronary artery diseases.
 4. The methodof claim 1, wherein said tissue to be promoted for angiogenesis isimplicated in an ischemic disease.
 5. The method of claim 1, whereinsaid angiogenesis promotion is implicated in wound healing.
 6. Themethod of claim 1, wherein said angiogenesis promotion is implicated inprevention of scar formation.
 7. The method of claim 3, furthercomprises directly contacting said tissue with said effective amount ofsaid Angelica sinensis extract SBD.4 or a fraction or a lyophilizatethereof, or one or more active components contained in said extract, incombination with a growth factor selected from the group consisting offibroblast growth factors (FGF), FGF-1, FGF-2, FGF-4, thymosins,platelet-derived growth factors (PDGF), insulin-binding growth factors(IGF), IGF-1, IGF-2, epidermal growth factor (EGF), transforming growthfactor (TGF), TGF-.alpha., TGF-.beta., cartilage inducing factors-A and-B, osteoid-inducing factors, osteogenin, bone morphogenic proteins, andother bone growth factors, collagen growth factors, heparin-bindinggrowth factor-1 or -2, and their biologically active derivatives.
 8. Themethod of claim 5, further comprises directly contacting said tissuewith said effective amount of said Angelica sinensis extract SBD.4 or afraction or a lyophilizate thereof, or one or more active componentscontained in said extract, in combination with collagen.
 9. The methodof claim 5, further comprises directly contacting said tissue with saideffective amount of said Angelica sinensis extract SBD.4 or a fractionor a lyophilizate thereof, or one or more active components contained insaid extract, in combination with polysaccharides.
 10. The method ofclaim 5, further comprises directly contacting said tissue with saideffective amount of said Angelica sinensis extract SBD.4 or a fractionor a lyophilizate thereof, or one or more active components contained insaid extract, in combination with skin substitutes.
 11. The method ofclaim 5, further comprises directly contacting said tissue with saideffective amount of said Angelica sinensis extract SBD.4 or a fractionor a lyophilizate thereof, or one or more active components contained insaid extract, in combination with antibiotics or other antimicrobialagents.
 12. The method in claim 3, wherein effective amount of saidAngelica sinensis extract SBD.4 or a fraction or a lyophilizate thereof,or one or more active components contained in said extract is formulatedin an edible power bar.
 13. The method of claim 3, further comprisesdirectly contacting said tissue with a pharmaceutically acceptableformulation of Angelica sinensis extract SBD.4 or a fraction or alyophilizate thereof, or one or more active components contained in saidextract.
 14. The formulation of claim 9, wherein said formulation ispackaged in a sachet.
 15. The formulation of claim 9, wherein saidformulation is a potable liquid.
 16. The formulation of claim 9, whereinsaid formulation is a tablet or pill.
 17. The formulation of claim 9,wherein said formulation is injectable.
 18. The method of claim 5,further comprising a method of treating a wound with a dressingmaterial, comprising: forming a gel with a gel forming amount of a watersoluble or water-swellable pharmaceutically acceptable polymer,excipients and effective amount of Angelica sinensis extract SBD.4 or afraction or a lyophilizate thereof, or one or more active componentscontained in said extract; dehydrating the gel to a preparation in theform of a foam or powder form of the gel material; attaching saidpreparation to a backing; said backing containing said preparation beingapplied to the wound.
 19. The method of claim 18, wherein said foam orpowder is in a suitable form so that it will adhere to a wound for atleast 24 hours and during that time, enhance healing of the wound andhelp prevent infection of the wound.
 20. The method of claim 18 whereinthe backing is an adhesive medical tape.
 21. The method of claim 18wherein the backing comprises a porous material.
 22. The method of claim18 wherein the backing comprises a non-porous material.
 23. The methodof claim 18 wherein the backing comprises a micro-porous material. 24.The method of claim 18 wherein the preparation is partially rehydratedto form a hydro-gel prior to application to the wound.
 25. The method ofclaim 5, further comprising a method of treating a wounded and otherwisedamaged skin with an aqueous soaking solution containing SBD.4
 26. Anacceptable formulation for cosmetic use as lotion, cream, cleanser andshampoo comprising effective amount of Angelica sinensis extract SBD.4or a fraction or a lyophilizate thereof.
 27. The formulation of claim26, wherein said formulation contains a skin-soothing agent selectedfrom the group consisting of dimethicone and allantoin.
 28. Theformulation of claim 26, wherein said formulation contains a UVscreening agent and a whitening agent.
 29. A method of promotingfibroblast growth in a mammalian tissue comprising contacting saidtissue with an effective amount of Angelica sinensis extract SBD.4 or afraction or a lyophilizate thereof, or one or more active componentscontained in said extract, to promote wound-healing in said tissue. 30.The method of claim 29, wherein said fibroblast growth promotion isassociated with increased collagen output.
 31. The method of claim 29,wherein said fibroblast growth promotion is implicated in wound healing.32. The method of claim 29, wherein said increased collagen output isimplicated in wound healing.
 33. The method of claim 29, wherein saidAngelica sinensis extract SBD.4 or a fraction or a lyophilizate thereof,or one or more active components contained in said extract, containscomponents selected from the group consisting of molecules withmass-to-charge (M/Z) ratio value of 147, 174, 218,216, 214, 304, 193,250,307 and
 406. 34. The method of claim 33, wherein said activecomponent is selected from the group consisting of molecules withmass-to-charge (M/Z) ratio value of
 174. 35. The method of claim 33,wherein said active component is a molecule with mass-to-charge (M/Z)ratio value of
 218. 36. The method of claim 26, wherein said Angelicasinensis extract SBD.4 or a fraction or a lyophilizate thereof, or oneor more active components contained in said extract, contains componentsselected from the group consisting of molecules with mass-to-charge(M/Z) ratio value of 147, 174, 218, 216, 214,304, 193, 250,307 and 406.37. The method of claim 36, wherein said active component is a moleculewith mass-to-charge (M/Z) ratio value of
 174. 38. The method of claim36, wherein said active component is a molecule with mass-to-charge(M/Z) ration value of
 218. 39. The method of claim 1, wherein saidAngelica sinensis extract SBD.4 or a fraction or a lyophilizate thereof,or one or more active components contained in said extract, containscomponents selected from the group consisting of molecules withmass-to-charge (M/Z) ratio value of 147, 174, 218, 216, 214, 304, 193,250, 307 and
 406. 40. The method of claim 39, wherein said activecomponent is a molecule with mass-to-charge (M/Z) ratio value of 1 74.41. The method of claim 39, wherein said active component is a moleculewith mass-to-charge (M/Z) ratio value of 218.